Molecular insights into the prototypical single-stranded DNA-binding protein from E. coli.

The SSB protein of Escherichia coli functions to bind single-stranded DNA wherever it occurs during DNA metabolism. Depending upon conditions, SSB occurs in several different binding modes. In the course of its function, SSB diffuses on ssDNA and transfers rapidly between different segments of ssDNA. SSB interacts with many other proteins involved in DNA metabolism, with 22 such SSB-interacting proteins, or SIPs, defined to date. These interactions chiefly involve the disordered and conserved C-terminal residues of SSB. When not bound to ssDNA, SSB can aggregate to form a phase-separated biomolecular condensate. Current understanding of the properties of SSB and the functional significance of its many intermolecular interactions are summarized in this review.

Biochemical characterization of Escherichia coli DnaC variants that alter DnaB helicase loading onto DNA.

DNA replication in Escherichia coli starts with loading of the replicative helicase, DnaB, onto DNA. This reaction requires the DnaC loader protein, which forms a 6:6 complex with DnaB and opens a channel in the DnaB hexamer through which single-stranded DNA is thought to pass. During replication, replisomes frequently encounter DNA damage and nucleoprotein complexes that can lead to replication fork collapse. Such events require DnaB re-loading onto DNA to allow replication to continue. Replication restart proteins mediate this process by recruiting DnaB6/DnaC6 to abandoned DNA replication forks. Several dnaC mutations that bypass the requirement for replication restart proteins or that block replication restart have been identified in E. coli. To better understand how these DnaC variants function, we have purified and characterized the protein products of several such alleles. Unlike wild-type DnaC, three of the variants (DnaC 809, DnaC 809,820, and DnaC 811) can load DnaB onto replication forks bound by single-stranded DNA-binding protein. DnaC 809 can also load DnaB onto double-stranded DNA. These results suggest that structural changes in the variant DnaB6/DnaC6 complexes expand the range of DNA substrates that can be used for DnaB loading, obviating the need for the existing replication restart pathways. The protein product of dnaC1331, which phenocopies deletion of the priB replication restart gene, blocks loading through the major restart pathway in vitro. Overall, the results of our study highlight the utility of bacterial DnaC variants as tools for probing the regulatory mechanisms that govern replicative helicase loading.

Interaction with the carboxy-terminal tip of SSB is critical for RecG function in E. coli.

In Escherichia coli, the single-stranded DNA-binding protein (SSB) acts as a genome maintenance organizational hub by interacting with multiple DNA metabolism proteins. Many SSB-interacting proteins (SIPs) form complexes with SSB by docking onto its carboxy-terminal tip (SSB-Ct). An alternative interaction mode in which SIPs bind to PxxP motifs within an intrinsically-disordered linker (IDL) in SSB has been proposed for the RecG DNA helicase and other SIPs. Here, RecG binding to SSB and SSB peptides was measured in vitro and the RecG/SSB interface was identified. The results show that RecG binds directly and specifically to the SSB-Ct, and not the IDL, through an evolutionarily conserved binding site in the RecG helicase domain. Mutations that block RecG binding to SSB sensitize E. coli to DNA damaging agents and induce the SOS DNA-damage response, indicating formation of the RecG/SSB complex is important in vivo. The broader role of the SSB IDL is also investigated. E. coli ssb mutant strains encoding SSB IDL deletion variants lacking all PxxP motifs retain wildtype growth and DNA repair properties, demonstrating that the SSB PxxP motifs are not major contributors to SSB cellular functions.

G-quadruplexes in bacteria: insights into the regulatory roles and interacting proteins of non-canonical nucleic acid structures.

G-quadruplexes (G4s) are highly stable, non-canonical DNA or RNA structures that can form in guanine-rich stretches of nucleic acids. G4-forming sequences have been found in all domains of life, and proteins that bind and/or resolve G4s have been discovered in both bacterial and eukaryotic organisms. G4s regulate a variety of cellular processes through inhibitory or stimulatory roles that depend upon their positions within genomes or transcripts. These include potential roles as impediments to genome replication, transcription, and translation or, in other contexts, as activators of genome stability, transcription, and recombination. This duality suggests that G4 sequences can aid cellular processes but that their presence can also be problematic. Despite their documented importance in bacterial species, G4s remain understudied in bacteria relative to eukaryotes. In this review, we highlight the roles of bacterial G4s by discussing their prevalence in bacterial genomes, the proteins that bind and unwind G4s in bacteria, and the processes regulated by bacterial G4s. We identify limitations in our current understanding of the functions of G4s in bacteria and describe new avenues for studying these remarkable nucleic acid structures.

The intrinsically disordered linker in the single-stranded DNA-binding protein influences DNA replication restart and recombination pathways in Escherichia coli K-12.

Tetrameric single-stranded (ss) DNA-binding proteins (SSBs) stabilize ssDNA intermediates formed during genome maintenance reactions in Bacteria. SSBs also recruit proteins important for these processes through direct SSB-protein interactions, including proteins involved in DNA replication restart and recombination processes. SSBs are composed of an N-terminal oligomerization and ssDNA-binding domain, a C-terminal acidic tip that mediates SSB-protein interactions, and an internal intrinsically disordered linker (IDL). Deletions and insertions into the IDL are well tolerated with few phenotypes, although the largest deletions and insertions exhibit some sensitivity to DNA-damaging agents. To define specific DNA metabolism processes dependent on IDL length, ssb mutants that lack 16, 26, 37, or 47 residues of the 57-residue IDL were tested for synthetic phenotypes with mutations in DNA replication restart or recombination genes. We also tested the impact of integrating a fluorescent domain within the SSB IDL using an ssb::mTur2 insertion mutation. Only the largest deletion tested or the insertion mutation causes sensitivity in any of the pathways. Mutations in two replication restart pathways (PriA-B1 and PriA-C) showed synthetic lethalities or small colony phenotypes with the largest deletion or insertion mutations. Recombination gene mutations del(recBCD) and del(ruvABC) show synthetic phenotypes only when combined with the largest ssb deletion. These results suggest that a minimum IDL length is important in some genome maintenance reactions in Escherichia coli. These include pathways involving PriA-PriB1, PriA-PriC, RecFOR, and RecG. The mTur2 insertion in the IDL may also affect SSB interactions in some processes, particularly the PriA-PriB1 and PriA-PriC replication restart pathways.IMPORTANCEssb is essential in Escherichia coli due to its roles in protecting ssDNA and coordinating genome maintenance events. While the DNA-binding core and acidic tip have well-characterized functions, the purpose of the intrinsically disordered linker (IDL) is poorly understood. In vitro studies have revealed that the IDL is important for cooperative ssDNA binding and phase separation. However, single-stranded (ss) DNA-binding protein (SSB) variants with large deletions and insertions in the IDL support normal cell growth. We find that the PriA-PriB1 and PriA-C replication restart, as well as the RecFOR- and RecG-dependent recombination, pathways are sensitive to IDL length. This suggests that cooperativity, phase separation, or a longer spacer between the core and acidic tip of SSB may be important for specific cellular functions.

Interaction with single-stranded DNA-binding protein modulates Escherichia coli RadD DNA repair activities.

The bacterial RadD enzyme is important for multiple genome maintenance pathways, including RecA DNA strand exchange and RecA-independent suppression of DNA crossover template switching. However, much remains unknown about the precise roles of RadD. One potential clue into RadD mechanisms is its direct interaction with the single-stranded DNA binding protein (SSB), which coats single-stranded DNA exposed during genome maintenance reactions in cells. Interaction with SSB stimulates the ATPase activity of RadD. To probe the mechanism and importance of RadD-SSB complex formation, we identified a pocket on RadD that is essential for binding SSB. In a mechanism shared with many other SSB-interacting proteins, RadD uses a hydrophobic pocket framed by basic residues to bind the C-terminal end of SSB. We found that RadD variants that substitute acidic residues for basic residues in the SSB binding site impair RadD:SSB complex formation and eliminate SSB stimulation of RadD ATPase activity in vitro. Additionally, mutant Escherichia coli strains carrying charge reversal radD changes display increased sensitivity to DNA damaging agents synergistically with deletions of radA and recG, although the phenotypes of the SSB-binding radD mutants are not as severe as a full radD deletion. This suggests that cellular RadD requires an intact interaction with SSB for full RadD function.

Wastewater Surveillance for Identifying SARS-CoV-2 Infections in Long-Term Care Facilities, Kentucky, USA, 2021-2022.

Persons living in long-term care facilities (LTCFs) were disproportionately affected by COVID-19. We used wastewater surveillance to detect SARS-CoV-2 infection in this setting by collecting and testing 24-hour composite wastewater samples 2-4 times weekly at 6 LTCFs in Kentucky, USA, during March 2021-February 2022. The LTCFs routinely tested staff and symptomatic and exposed residents for SARS-CoV-2 using rapid antigen tests. Of 780 wastewater samples analyzed, 22% (n = 173) had detectable SARS-CoV-2 RNA. The LTCFs reported 161 positive (of 16,905) SARS-CoV-2 clinical tests. The wastewater SARS-CoV-2 signal showed variable correlation with clinical test data; we observed the strongest correlations in the LTCFs with the most positive clinical tests (n = 45 and n = 58). Wastewater surveillance was 48% sensitive and 80% specific in identifying SARS-CoV-2 infections found on clinical testing, which was limited by frequency, coverage, and rapid antigen test performance.

Simultaneous evaluation of treatment efficacy and toxicity for bispecific T-cell engager therapeutics in a humanized mouse model.

Immuno-oncology (IO)-based therapies such as checkpoint inhibitors, bi-specific antibodies, and CAR-T-cell therapies have shown significant success in the treatment of several cancer indications. However, these therapies can result in the development of severe adverse events, including cytokine release syndrome (CRS). Currently, there is a paucity of in vivo models that can evaluate dose-response relationships for both tumor control and CRS-related safety issues. We tested an in vivo PBMC humanized mouse model to assess both treatment efficacy against specific tumors and the concurrent cytokine release profiles for individual human donors after treatment with a CD19xCD3 bispecific T-cell engager (BiTE). Using this model, we evaluated tumor burden, T-cell activation, and cytokine release in response to bispecific T-cell-engaging antibody in humanized mice generated with different PBMC donors. The results show that PBMC engrafted NOD-scid Il2rgnull mice lacking expression of mouse MHC class I and II (NSG-MHC-DKO mice) and implanted with a tumor xenograft predict both efficacy for tumor control by CD19xCD3 BiTE and stimulated cytokine release. Moreover, our findings indicate that this PBMC-engrafted model captures variability among donors for tumor control and cytokine release following treatment. Tumor control and cytokine release were reproducible for the same PBMC donor in separate experiments. The PBMC humanized mouse model described here is a sensitive and reproducible platform that identifies specific patient/cancer/therapy combinations for treatment efficacy and development of complications.

Mismatch discrimination and sequence bias during end-joining by DNA ligases.

DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3'-OH and 5'-phosphorylated ends in DNA. To determine whether DNA annealing equilibria or properties intrinsic to the DNA ligase enzyme impact end-joining ligation outcomes, we used a highly multiplexed, sequencing-based assay to profile mismatch discrimination and sequence bias for several ligases capable of efficient end-joining. Our data reveal a spectrum of fidelity and bias, influenced by both the strength of overhang annealing as well as sequence preferences and mismatch tolerances that vary both in degree and kind between ligases. For example, while T7 DNA ligase shows a strong preference for ligating high GC sequences, other ligases show little GC-dependent bias, with human DNA Ligase 3 showing almost none. Similarly, mismatch tolerance varies widely among ligases, and while all ligases tested were most permissive of G:T mismatches, some ligases also tolerated bulkier purine:purine mismatches. These comprehensive fidelity and bias profiles provide insight into the biology of end-joining reactions and highlight the importance of ligase choice in application design.

Identification of recG genetic interactions in Escherichia coli by transposon sequencing.

IMPORTANCE: DNA damage and subsequent DNA repair processes are mutagenic in nature and an important driver of evolution in prokaryotes, including antibiotic resistance development. Genetic screening approaches, such as transposon sequencing (Tn-seq), have provided important new insights into gene function and genetic relationships. Here, we employed Tn-seq to gain insight into the function of the recG gene, which renders Escherichia coli cells moderately sensitive to a variety of DNA-damaging agents when they are absent. The reported recG genetic interactions can be used in combination with future screens to aid in a more complete reconstruction of DNA repair pathways in bacteria.

Enhanced development of functional human NK cells in NOD-scid-IL2rgnull mice expressing human IL15.

Human innate immunity plays a critical role in tumor surveillance and in immunoregulation within the tumor microenvironment. Natural killer (NK) cells are innate lymphoid cells that have opposing roles in the tumor microenvironment, including NK cell subsets that mediate tumor cell cytotoxicity and subsets with regulatory function that contribute to the tumor immune suppressive environment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function in tumorigenesis. Humanized mice are a powerful alternative to syngeneic mouse tumor models for the study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However, human NK cell development and survival in humanized NOD-scid-IL2rgnull (NSG) mice are severely limited. To enhance NK cell development, we have developed NSG mice that constitutively expresses human Interleukin 15 (IL15), NSG-Tg(Hu-IL15). Following hematopoietic stem cell engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of functional human CD56+ NK cells are detectable in blood and spleen, as compared to NSG mice. Hematopoietic stem cell (HSC)-engrafted NSG-Tg(Hu-IL15) mice also supported the development of human CD3+ T cells, CD20+ B cells, and CD33+ myeloid cells. Moreover, the growth kinetics of a patient-derived xenograft (PDX) melanoma were significantly delayed in HSC-engrafted NSG-Tg(Hu-IL15) mice as compared to HSC-engrafted NSG mice demonstrating that human NK cells have a key role in limiting the tumor growth. Together, these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells, which limit the growth of PDX tumors.

Evaluating Scalable Supervised Learning for Synthesize-on-Demand Chemical Libraries.

Traditional small-molecule drug discovery is a time-consuming and costly endeavor. High-throughput chemical screening can only assess a tiny fraction of drug-like chemical space. The strong predictive power of modern machine-learning methods for virtual chemical screening enables training models on known active and inactive compounds and extrapolating to much larger chemical libraries. However, there has been limited experimental validation of these methods in practical applications on large commercially available or synthesize-on-demand chemical libraries. Through a prospective evaluation with the bacterial protein-protein interaction PriA-SSB, we demonstrate that ligand-based virtual screening can identify many active compounds in large commercial libraries. We use cross-validation to compare different types of supervised learning models and select a random forest (RF) classifier as the best model for this target. When predicting the activity of more than 8 million compounds from Aldrich Market Select, the RF substantially outperforms a naïve baseline based on chemical structure similarity. 48% of the RF's 701 selected compounds are active. The RF model easily scales to score one billion compounds from the synthesize-on-demand Enamine REAL database. We tested 68 chemically diverse top predictions from Enamine REAL and observed 31 hits (46%), including one with an IC50 value of 1.3 µM.

Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp.

The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here, we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK), revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A survey of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria, where (p)ppGpp regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.

Establishment and initial implementation of the Australasian Pelvic Floor Procedure Registry.

INTRODUCTION AND HYPOTHESIS: Stress urinary incontinence (SUI) and pelvic organ prolapse (POP) are common pelvic floor disorders (PFDs). Owing to significant adverse events associated with mesh-related pelvic floor procedures (PFPs) in a proportion of the surgically treated population, and deficits in collection and reporting of these events, the Australian Government identified an urgent need for a tracking mechanism to improve safety and quality of care. The Australasian Pelvic Floor Procedure Registry (APFPR) was recently established following the 2018 Senate Committee Inquiry with the aim of tracking outcomes of PFP involving the use of devices and/or prostheses, with the objective of improving the health outcomes of women who undergo these procedures. This paper will describe the APFPR's aims, development, implementation and possible challenges on the way to its establishment. METHODS: The APFPR has been developed and implemented in accordance with the national operating principles of clinical quality registries (CQRs). The minimum datasets (MDS) for the registry's database have been developed using a modified Delphi process, and data are primarily being collected from participating surgeons. Patient recruitment is based on an opt-out approach or a waiver of consent. Patient-reported outcome measures (PROMs) providing additional health and outcome information will be obtained from participating women to support safety monitoring of mesh-related adverse events. RESULTS: Currently in the Australasian Pelvic Floor Procedure Registry (APFPR) there are 32 sites from various jurisdictions across Australia, that have obtained relevant ethics and governance approvals to start patient recruitment and data collection as of January 2023. Additionally, there are two sites that are awaiting governance review and five sites that are having documentation compiled for submission. Seventeen sites have commenced patient registration and have entered data into the database. Thus far, we have 308 patients registered in the APFPR database. The registry also published its first status report and a consumer-friendly public report in 2022. CONCLUSIONS: The registry will act as a systematic tracking mechanism by collecting outcomes on PFP, especially those involving devices and/or prostheses to improve safety and quality of care.

Have changing practices in salvage medical options affected colectomy rates in acute severe ulcerative colitis?

BACKGROUND: In 2014, infliximab (IFX) was listed on the Australian Pharmaceutical Benefits Scheme for acute severe ulcerative colitis (ASUC) and is now the preferred option for medical salvage, superseding cyclosporin A (CsA). Optimal dosing schedules for IFX remain unknown. AIM: The authors aim to evaluate the effect of changing from predominantly CsA to almost exclusively IFX for the treatment of steroid-refractory ASUC on colectomy rates. METHODS: A retrospective review was performed of patients admitted with ASUC between 2012 and 2020. Patients were categorised into two groups according to year of presentation - either 'historical treatment' cohort (2012-2014), when CsA was primarily used, or 'contemporary treatment' cohort (2014-2020), when IFX was mostly prescribed, in either standard or intensive doses. RESULTS: One hundred thirty-nine patients were included; 37 in the historical treatment cohort and 102 in the contemporary treatment cohort. In the historical treatment cohort, 12 of 37 received salvage therapy and eight (67%) received CsA. In the contemporary treatment cohort, 49 of 102 patients received salvage therapy, 40 (82%) with IFX, of whom 22 (53%) received intensified doses. Colectomy rates were similar at 30 days, 6 months and 12 months between historical and contemporary treatment cohorts (14% vs 12% [P = 0.77], 19% vs 18% [P > 0.99],and 22% vs 18% [P = 0.63], respectively). Difference in 12-month colectomy rates between standard versus intensive IFX did not meet statistical significance (three of 21 [14%] vs nine of 22 [41%]. respectively; P = 0.09). CONCLUSION: There was no difference in 30-day, 6-month or 12-month colectomy rates between the historical treatment and contemporary treatment cohorts. The use of IFX, rather than CsA, even at intensified dosing, does not appear to reduce the colectomy rate observed in our patients.

Natural Transformation Protein ComFA Exhibits Single-Stranded DNA Translocase Activity.

Natural transformation is one of the major mechanisms of horizontal gene transfer in bacterial populations and has been demonstrated in numerous species of bacteria. Despite the prevalence of natural transformation, much of the molecular mechanism remains unexplored. One major outstanding question is how the cell powers DNA import, which is rapid and highly processive. ComFA is one of a few proteins required for natural transformation in Gram-positive bacteria. Its structural resemblance to the DEAD box helicase family has led to a long-held hypothesis that ComFA acts as a motor to help drive DNA import into the cytosol. Here, we explored the helicase and translocase activity of ComFA to address this hypothesis. We followed the DNA-dependent ATPase activity of ComFA and, combined with mathematical modeling, demonstrated that ComFA likely translocates on single-stranded DNA from 5' to 3'. However, this translocase activity does not lead to DNA unwinding under the conditions we tested. Further, we analyzed the ATPase cycle of ComFA and found that ATP hydrolysis stimulates the release of DNA, providing a potential mechanism for translocation. These findings help define the molecular contribution of ComFA to natural transformation and support the conclusion that ComFA plays a key role in powering DNA uptake. IMPORTANCE Competence, or the ability of bacteria to take up and incorporate foreign DNA in a process called natural transformation, is common in the bacterial kingdom. Research in several bacterial species suggests that long, contiguous stretches of DNA are imported into cells in a processive manner, but how bacteria power transformation remains unclear. Our finding that ComFA, a DEAD box helicase required for competence in Gram-positive bacteria, translocates on single-stranded DNA from 5' to 3', supports the long-held hypothesis that ComFA may be the motor powering DNA transport during natural transformation. Moreover, ComFA may be a previously unidentified type of DEAD box helicase-one with the capability of extended translocation on single-stranded DNA.

Escherichia coli K-12 has two distinguishable PriA-PriB replication restart pathways.

In Escherichia coli, PriA, PriB, PriC, and DnaT proteins mediate three pathways for Replication Restart called PriA-PriB, PriA-PriC, and PriC. PriA is crucial for two of the three pathways. Its absence leads to slow growth, high basal levels of SOS expression, poorly partitioning nucleoids, UV sensitivity, and recombination deficiency. PriA has ATPase and helicase activities and interacts with PriB, DnaT, and single-stranded DNA-binding protein (SSB). priA300 (K230R) and priA301 (C479Y) have no phenotype as single mutants, but each phenocopy a priA-null mutant combined with ∆priB. This suggested that the two priA mutations affected the helicase activity that is required for the PriA-PriC pathway. To further test this, the biochemical activities of purified PriA300 and PriA301 were examined. As expected, PriA300 lacks ATPase and helicase activities but retains the ability to interact with PriB. PriA301, however, retains significant PriB-stimulated helicase activity even though PriA301 interactions with PriB and DNA are weakened. A PriA300,301 variant retains only the ability to interact with DNA in vitro and phenocopies the priA-null phenotype in vivo. This suggests that there are two biochemically and genetically distinct PriA-PriB pathways. One uses PriB-stimulated helicase activity to free a region of ssDNA and the other uses helicase-independent remodeling activity.

Pudendal Nerve Block for Posthemorrhoidectomy Pain: A Prospective, Single-Blinded Randomized Control Trial.

BACKGROUND: Postoperative pain is common in hemorrhoidectomy patients. Local anesthetic given either as an intraoperative pudendal nerve block or as a local wound infiltration may help alleviate postoperative pain. OBJECTIVES: This study sought to determine whether the addition of an intraoperative pudendal nerve block to a perianal local wound infiltration and standardized analgesia regimen was superior to a perianal local wound infiltration and standardized analgesia regimen alone in reducing early postoperative pain following hemorrhoidectomy. The secondary objective was to measure differences between treatment groups in perceived perianal numbness, oral opioid requirements, and adverse events. DESIGN: This study was a prospective, single-blinded randomized controlled trial approved by the Eastern Health Human Research and Ethics Committee in Melbourne, Australia (registration number: E09/2014). SETTINGS: Patients were recruited across 3 Australian hospitals. PATIENTS: Eighty patients with symptomatic hemorrhoids requiring hemorrhoidectomy in colorectal surgical outpatient clinics were successfully recruited and enrolled in the study, with 1 patient later dropping out. INTERVENTION: Patients were randomly assigned to either the pudendal nerve block group or a control group. The pudendal nerve block group received 5 mL bupivacaine 0.5% with adrenaline 1:200,000 to both pudendal nerve trunks bilaterally. Both groups received 10 mL of the same local anesthetic injected into the post-hemorrhoidectomy wound bed. MAIN OUTCOME MEASURES: Visual analogue scales were used to record patient pain scores. Dichotomous (yes/no) answers were recorded for secondary objectives. RESULTS: There were no significant differences in postoperative pain between groups at 4 hours, 8 hours, 12 hours, or 24 hours. Additionally, there were no significant differences between groups with respect to perceived perianal numbness, oral opioid usage or adverse events. LIMITATIONS: The authors recognize that without a nerve stimulator, an argument can be made that the pudendal nerve block was not actually achieved. CONCLUSION: Pudendal nerve block does not appear to demonstrate additional benefit in post-hemorrhoidectomy pain reduction beyond local anesthetic to the wound. See Video Abstract at http://links.lww.com/DCR/B780. BLOQUEO DEL NERVIO PUDENDO PARA EL DOLOR POSHEMORROIDECTOMA ESTUDIO PROSPECTIVO, ALEATORIO, CONTROLADO, CIEGO: ANTECEDENTES:El dolor posoperatorio es común en pacientes luego de una hemorroidectomía. La administración de anestésico local como bloqueo del nervio pudendo intraoperatorio o infiltración local de la herida puede ayudar a aliviar el dolor posoperatorio.OBJETIVOS:Determinar si agregar un bloqueo intraoperatorio del nervio pudendo a una infiltración local perianal de la herida y un régimen de analgesia estandarizado fue superior a una infiltración local perianal de la herida más un régimen de analgesia estandarizado para reducir el dolor posoperatorio precoz después de una hemorroidectomía. Los objetivos secundarios incluyeron sensación de adormecimiento perianal, requerimientos de opioides orales y eventos adversos informados.DISEÑO:Este estudio fue un ensayo controlado aleatorio, prospectivo, ciego, aprobado por el Comité de Ética e Investigación en Humanos de Eastern Health en Melbourne, Australia (número de registro: E09 / 2014).ESCENARIO:Los pacientes fueron reclutados en tres hospitales australianos.PACIENTES:Ochenta pacientes se inscribieron con éxito en el estudio, y más tarde un paciente abandonó.INTERVENCIÓN:Los pacientes fueron asignados al azar al grupo de bloqueo del nervio pudendo o al grupo control. El grupo de bloqueo del nervio pudendo recibió 5 ml de bupivacaína al 0,5% con adrenalina 1: 200.000 en ambos troncos del nervio pudendo bilateralmente. Ambos grupos recibieron 10 ml del mismo anestésico local inyectado en el lecho de la herida posterior a la hemorroidectomía.PRINCIPALES VARIABLES ANALIZADAS:Se utilizaron escalas analógicas visuales para registrar las puntuaciones de dolor del paciente. Se registraron respuestas dicotómicas (sí / no) para los objetivos secundarios.RESULTADOS:No hubo diferencias significativas en el dolor posoperatorio entre los grupos a las 4, 8, 12 o 24 horas. Además, no hubo diferencias significativas entre los grupos con respecto al adromecimiento perianal percibido, el uso de opioides orales o los eventos adversos.LIMITACIONES:Sin el uso de un estimulador nervioso, se puede argumentar que el bloqueo del nervio pudendo no se logró realmente.CONCLUSIÓNES:El bloqueo del nervio pudendo no parece demostrar un beneficio adicional en la reducción del dolor posterior a la hemorroidectomía más allá del anestésico local en la herida. Consulte Video Resumen en http://links.lww.com/DCR/B780.

E. coli Rep helicase and RecA recombinase unwind G4 DNA and are important for resistance to G4-stabilizing ligands.

G-quadruplex (G4) DNA structures can form physical barriers within the genome that must be unwound to ensure cellular genomic integrity. Here, we report unanticipated roles for the Escherichia coli Rep helicase and RecA recombinase in tolerating toxicity induced by G4-stabilizing ligands in vivo. We demonstrate that Rep and Rep-X (an enhanced version of Rep) display G4 unwinding activities in vitro that are significantly higher than the closely related UvrD helicase. G4 unwinding mediated by Rep involves repetitive cycles of G4 unfolding and refolding fueled by ATP hydrolysis. Rep-X and Rep also dislodge G4-stabilizing ligands, in agreement with our in vivo G4-ligand sensitivity result. We further demonstrate that RecA filaments disrupt G4 structures and remove G4 ligands in vitro, consistent with its role in countering cellular toxicity of G4-stabilizing ligands. Together, our study reveals novel genome caretaking functions for Rep and RecA in resolving deleterious G4 structures.

Development of a single-stranded DNA-binding protein fluorescent fusion toolbox.

Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.